Application of cinobufotalin in preparing a drug for treating tuberous sclerosis complex-related renal angiomyolipoma

Abstract

The present invention belongs to the field of biomedical technologies, and particularly relates to the application of cinobufotalin in the preparation of drugs for treating tuberous sclerosis complex-related renal angiomyolipoma. The cinobufotalin can directly inhibit the growth of tuberous sclerosis complex-related renal angiomyolipoma cells and increase the protein expression level of apoptosis markers in tuberous sclerosis complex-related renal angiomyolipoma cells. The cinobufotalin can be used to prepare drugs for treating tuberous sclerosis complex-related renal angiomyolipoma. At the same time, the cost of cinobufotalin is relatively low, providing a new effective and low-cost potential drug for the treatment of tuberous sclerosis complex-related renal angiomyolipoma.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of cinobufolin in the preparation of a drug for resisting tuberous sclerosis complex-related renal angiomyolipoma. Background Art

[0002] Tuberous sclerosis complex-associated renal angiomyolipoma (TSC-RAML) is a benign renal tumor composed of blood vessels, smooth muscle and adipose tissue. Its etiology is closely related to genetics. Mutations or abnormal expressions of specific genes TSC1 / 2 activate the m-Tor pathway, affecting the growth, differentiation and proliferation of renal cells, leading to abnormal proliferation of tissue components and formation of hamartomas. Endocrine disorders and cytokine network imbalance are also one of the causes of tuberous sclerosis complex-associated renal angiomyolipoma. For example, estrogen changes affect the renal microenvironment, and cytokine interactions interfere with cell signal transduction, causing an imbalance in the proliferation and apoptosis of renal cells, which will form tuberous sclerosis complex-associated renal angiomyolipoma.

[0003] Treatment of tuberous sclerosis complex-related renal angiomyolipoma depends on tumor size, symptoms, and the patient's physical condition. Smaller asymptomatic tumors can be followed up regularly, and growth can be monitored using ultrasound, CT, or MRI. If the tumor diameter exceeds the standard or symptoms occur, such as low back pain, hematuria, and abdominal mass compression, the treatment is mainly surgery and drug therapy. mTOR inhibitors are key drugs for the treatment of tuberous sclerosis complex-related renal angiomyolipoma, acting on mTOR to inhibit its activity, block signaling pathways, inhibit tumor cell proliferation, induce apoptosis, and inhibit angiogenesis.

[0004] However, existing methods for treating tuberous sclerosis complex-related renal angiomyolipoma all have defects. For example, surgery can remove the tumor, but it is very traumatic, and there may be bleeding, infection, and damage to renal function after surgery. It is difficult to remove tumors in special locations or multiple tumors, and it is easy to be incompletely removed or relapse. There are large individual differences in drug treatment responses. Some patients are effective, while others are insensitive. It is difficult to cure and it is easy to relapse after stopping the drug. Long-term use will also produce drug resistance and reduced efficacy. The high price puts a heavy financial burden on patients. It is difficult for those with poor financial conditions to persist. It is harmful to the fetus and is limited to special populations. Liver and kidney metabolism will increase the burden on the liver and kidneys. It needs to be used with caution or the dosage should be adjusted. Improvements or alternatives are urgently needed.

[0005] In summary, finding a more effective, safe, comprehensive and targeted treatment for tuberous sclerosis complex-related renal angiomyolipoma has important clinical significance and social value. Summary of the invention

[0006] Based on the above technical background, the main purpose of the present invention is to provide the use of cinobuczalin in the preparation of drugs against tuberous sclerosis complex-related renal angiomyolipoma, so as to overcome the shortcomings of the prior art.

[0007] To achieve the aforementioned invention object, the technical solution adopted by the present invention includes:

[0008] In a first aspect of the present invention, there is provided the use of cinobufotalin in the preparation of a drug for treating tuberous sclerosis complex-related renal angiomyolipoma.

[0009] Preferably, the chemical structural formula of the cinobufotalin is shown in formula (1):

[0010] Formula (1).

[0011] Preferably, the cinobufotalin can inhibit the growth of tuberous sclerosis complex-related renal angiomyolipoma cells.

[0012] Preferably, the cinobufotalin can increase the protein expression level of apoptosis markers in tuberous sclerosis complex-related renal angiomyolipoma cells.

[0013] More preferably, the cinobufotalin can increase the protein expression levels of Caspas3, Caspas9, Cytochrome C, and γ-H2A.X in tuberous sclerosis complex-related renal angiomyolipoma cells.

[0014] Preferably, the cinobufotalin can reduce the protein expression levels of CCNA2 and CDK2 in tuberous sclerosis complex-related renal angiomyolipoma cells, and the cinobufotalin can increase the protein expression level of p16 in tuberous sclerosis complex-related renal angiomyolipoma cells.

[0015] In a second aspect of the present invention, there is provided a drug for treating tuberous sclerosis complex-related renal angiomyolipoma, the active ingredient of the drug for treating tuberous sclerosis complex-related renal angiomyolipoma is cinobufotalin, and the drug for treating tuberous sclerosis complex-related renal angiomyolipoma further includes pharmaceutically acceptable excipients.

[0016] Preferably, the excipients include one or more of a sustained-release agent, a shaping agent, a solubilizer, a stabilizer, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, and a lubricant.

[0017] Preferably, the dosage form of the drug for treating tuberous sclerosis complex-related renal angiomyolipoma includes an oral preparation, a dressing preparation, an injection preparation, or an inhalation preparation.

[0018] The beneficial effects of the present invention:

[0019] (1)The present invention discovers that cinobufotalin can directly inhibit the growth of renal angiomyolipoma cells associated with tuberous sclerosis, and can increase the protein expression level of apoptosis markers in renal angiomyolipoma cells associated with tuberous sclerosis. Cinobufotalin can be used to prepare drugs for treating renal angiomyolipoma associated with tuberous sclerosis, providing a new potential drug for the treatment of renal angiomyolipoma associated with tuberous sclerosis.

[0020] (2)The present invention discovers that cinobufotalin can increase the protein expression levels of Caspas3, Caspas9, Cytochrome C, γ-H2A.X, and p16 in renal angiomyolipoma cells associated with tuberous sclerosis, and can reduce the protein expression levels of CCNA2 and CDK2 in renal angiomyolipoma cells associated with tuberous sclerosis, indicating that cinobufotalin can promote apoptosis of renal angiomyolipoma cells associated with tuberous sclerosis. Cinobufotalin can treat renal angiomyolipoma associated with tuberous sclerosis by inhibiting the growth of renal angiomyolipoma cells associated with tuberous sclerosis and promoting apoptosis of renal angiomyolipoma cells associated with tuberous sclerosis, and it has a good therapeutic and improvement effect on renal angiomyolipoma associated with tuberous sclerosis.

[0021] (3)Cinobufotalin is rich in sources. Applying it to drugs for treating renal angiomyolipoma associated with tuberous sclerosis can reduce the cost of drugs for treating renal angiomyolipoma associated with tuberous sclerosis, making the drugs for treating renal angiomyolipoma associated with tuberous sclerosis have both good curative effects and low costs, which has important clinical significance and social value for improving and treating renal angiomyolipoma associated with tuberous sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A bar graph showing the inhibitory effect of cinobufotalin on renal angiomyolipoma cells associated with tuberous sclerosis;

[0023] Figure 2 A bar graph showing the apoptotic effect of cinobufotalin on renal angiomyolipoma cells associated with tuberous sclerosis;

[0024] Figure 3 Showing the mechanism of cinobufotalin acting on cyclin kinases;

[0025] Figure 4 A schematic diagram showing the cell cycle arrest effect of cinobufotalin on renal angiomyolipoma cells associated with tuberous sclerosis;

[0026] Figure 5 A schematic diagram showing the quantitative analysis and statistical results of the Ct values of qPCR detection of related gene mRNAs. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will be described in detail below, and its features and advantages will become clearer and more definite with these descriptions.

[0028] The first aspect of the present invention lies in providing the use of cinobufotalin in the preparation of a drug for treating tuberous sclerosis complex-related renal angiomyolipoma.

[0029] The chemical structural formula of the cinobufotalin is shown as formula (1):

[0030] Formula (1).

[0031] The cinobufotalin has the effect of inhibiting the growth of tuberous sclerosis complex-related renal angiomyolipoma cells, that is, cinobufotalin can treat tuberous sclerosis complex-related renal angiomyolipoma by inhibiting the growth of tuberous sclerosis complex-related renal angiomyolipoma cells.

[0032] The cinobufotalin can increase the protein expression level of apoptosis markers in tuberous sclerosis complex-related renal angiomyolipoma cells.

[0033] Preferably, the cinobufotalin can increase the protein expression levels of Caspas3, Caspas9, Cytochrome C, and γ-H2A.X in tuberous sclerosis complex-related renal angiomyolipoma cells, and will result in a lower protein expression level of BCL-2.

[0034] The cinobufotalin can reduce the protein expression levels of CCNA2 and CDK2 in tuberous sclerosis complex-related renal angiomyolipoma cells, and the cinobufotalin can increase the protein expression level of p16 in tuberous sclerosis complex-related renal angiomyolipoma cells.

[0035] The cinobufotalin can promote the apoptosis of tuberous sclerosis complex-related renal angiomyolipoma cells, that is, cinobufotalin can treat tuberous sclerosis complex-related renal angiomyolipoma by promoting the apoptosis of tuberous sclerosis complex-related renal angiomyolipoma cells.

[0036] The active ingredient of the drug for treating tuberous sclerosis complex-related renal angiomyolipoma is cinobufotalin.

[0037] The second aspect of the present invention lies in providing a drug for treating tuberous sclerosis complex-related renal angiomyolipoma. The active ingredient of the drug for treating tuberous sclerosis complex-related renal angiomyolipoma is cinobufotalin, and the drug for treating tuberous sclerosis complex-related renal angiomyolipoma further includes pharmaceutically acceptable excipients.

[0038] Preferably, the adjuvants include one or more of a sustained-release agent, an excipient, a solubilizer, a stabilizer, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, and a lubricant.

[0039] Preferably, the dosage form of the drug for treating tuberous sclerosis complex-related renal angiomyolipoma includes an oral preparation, a dressing preparation, an injection preparation, or an inhalation preparation.

[0040] Preferably, the effective dose of cinobufotalin in animals for the drug for treating tuberous sclerosis complex-related renal angiomyolipoma is not higher than 10 mg / kg, and the effective dose in cells is not higher than 10 μM.

[0041] Examples

[0042] The present invention will be further illustrated by the following specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0043] Example 1 Cell Model

[0044] The primary TSC-RAML (tuberous sclerosis complex-related renal angiomyolipoma) cells (self-extracted from the laboratory) were inoculated into culture dishes and divided into a blank group and a cinobufotalin administration group.

[0045] The following operations were performed on the blank group (without adding cinobufotalin) and the cinobufotalin administration group respectively: After the primary cells grew well and the growth density was about 60%, the cinobufotalin administration group and the blank group were given the same volume of 1640 medium containing 10% serum and treated for 24 hours at the same time. The cinobufotalin administration group was evenly divided into 7 groups. After 24 hours of treatment, cinobufotalin solutions with concentrations of 0.1 μmol / mL, 0.3 μmol / mL, 0.5 μmol / mL, 1 μmol / mL, 3 μmol / mL, 5 μmol / mL, and 10 μmol / mL were added to the 7 groups of the cinobufotalin administration group respectively. The cell culture medium was aspirated, and 100 μL of CCK8 solution was added to each well and incubated for another 2 h. The test results of the inhibitory effects of the blank group and the cinobufotalin administration group on tuberous sclerosis complex-related renal angiomyolipoma cells are as Figure 1 shown.

[0046] From Figure 1Among them, compared with the blank group, cinobufotalin has an inhibitory effect on the proliferation of tuberous sclerosis complex-related renal angiomyolipoma cells. Moreover, with the increase in the concentration of cinobufotalin, its inhibitory effect on the proliferation of tuberous sclerosis complex-related renal angiomyolipoma cells gradually strengthens. When the concentration of cinobufotalin is 5 μmol / mL and 10 μmol / mL, the inhibitory effects of cinobufotalin on the proliferation of tuberous sclerosis complex-related renal angiomyolipoma cells are basically the same, and its inhibitory effect on the proliferation of tuberous sclerosis complex-related renal angiomyolipoma cells is the strongest. The above results indicate that cinobufotalin can inhibit the growth and proliferation of tuberous sclerosis complex-related renal angiomyolipoma cells. When the concentration of cinobufotalin is 5 μmol / mL, its inhibitory effect on the growth and proliferation of tuberous sclerosis complex-related renal angiomyolipoma cells is the strongest.

[0047] Example 2

[0048] Lyse and extract proteins from the blank group and the cinobufotalin administration groups (0.1 μmol / mL, 0.5 μmol / mL, 1 μmol / mL) in Example 1 using a protein lysis solution or use an RNA extraction reagent to extract proteins or mRNAs. Then, use immunoblotting (western blotting) or fluorescence quantitative PCR methods to detect the apoptosis markers (Caspas3, Caspas9, BCL-2, Cytochrome C, γ-H2A.X) and cell cycle markers (CCNA2, CDK2, p16) in each group. Extract proteins from TSC-RAML primary cells using 8M urea, add protease inhibitors (purchased from Beyotime Biotechnology Company) to the lysis solution, perform quantification using the BCA method, load equal amounts for polyacrylamide gel electrophoresis, use the internal reference protein actin for calibration, transfer the membrane after electrophoresis, block with 5% non-fat milk powder at room temperature for one hour, purchase the antibodies from Wuhan Aibote Biotechnology Co., Ltd. with a dilution concentration of 1:1000, incubate overnight with 1% non-fat milk powder, wash three times the next day, each time wash with 5% PBST5 for 5 minutes, then incubate with HRP-labeled secondary antibody, incubate with 1% non-fat milk powder at room temperature for one hour, wash three times, each time wash with 5% PBST5 for 5 minutes, use ECL reagent for luminescence, take and save photos for analysis. The detection results of the apoptosis markers are as Figure 2 shown, Figure 2 where the abscissa is the concentration of cinobufotalin, and the detection results of the cell cycle markers are as Figure 3 shown, Figure 3 where the abscissa is the concentration of cinobufotalin.

[0049] From Figure 2It can be seen that, compared with the blank group, the protein expression levels of Caspas3, Caspas9, Cytochrome C, and γ-H2A.X in the cinobufotalin group were higher, while the protein expression level of BCL-2 was lower. Moreover, as the concentration of cinobufotalin increased, the protein expression levels of Caspas3, Caspas9, Cytochrome C, and γ-H2A.X gradually increased. This indicates that cinobufotalin can increase the protein expression levels of apoptosis markers. Cinobufotalin can increase the protein expression levels of Caspas3, Caspas9, Cytochrome C, and γ-H2A.X in tuberous sclerosis-related renal angiomyolipoma cells, and as the concentration of cinobufotalin increases, its effect on the protein expression levels of apoptosis markers in tuberous sclerosis-related renal angiomyolipoma cells gradually increases, further indicating that cinobufotalin can promote the apoptosis of tuberous sclerosis-related renal angiomyolipoma cells.

[0050] It can be seen from Figure 3 that, compared with the blank group, the protein expression levels of CCNA2 and CDK2 in the cinobufotalin group decreased, while the protein expression level of p16 in the cinobufotalin group increased. Moreover, as the concentration of cinobufotalin increased, the protein expression levels of CCNA2 and CDK2 gradually decreased, and the protein expression level of p16 gradually increased. This indicates that cinobufotalin can decrease the protein expression levels of CCNA2 and CDK2 in tuberous sclerosis-related renal angiomyolipoma cells, and cinobufotalin can increase the protein expression level of p16 in tuberous sclerosis-related renal angiomyolipoma cells.

[0051] Example 3

[0052] Cell cycle flow cytometry analysis was performed on the blank group and the cinobufotalin administration groups (0.1 μmol / mL, 0.5 μmol / mL, 1 μmol / mL) in Example 1. The steps of cell cycle flow cytometry analysis are as follows: First, cells in the logarithmic growth phase were collected, digested with trypsin to make a single-cell suspension, and the cells were washed twice with PBS. Then, the cells were fixed in pre-cooled 70% ethanol and incubated overnight at 4°C to fix the DNA inside the cells. After that, the fixing solution ethanol was removed, and the cells were washed again with PBS. An appropriate amount of propidium iodide (PI) staining solution containing RNase A was added to make its final concentration capable of effective staining, and the cells were incubated in the dark at room temperature for about 30 minutes to allow PI to bind to the DNA inside the cells. Finally, the stained cell suspension was detected using a flow cytometer, and by analyzing the distribution of cells in different fluorescence intensity regions, the proportion of cells in each stage of the cell cycle was determined. The detection results are as Figure 4 shown. It can be seen from Figure 4It can be seen that cinobufotalin can arrest tuberous sclerosis complex-related renal angiomyolipoma cells in the G2 phase.

[0053] The RNA extraction reagent was used by the Trizol method. After the extracted RNA was quantified, reverse transcription was carried out (using a one-step reverse transcription kit purchased from Wuhan Aibotech Biotechnology Co., Ltd.). Then the cDNA was stored, primers were designed, and a fluorescence quantitative PCR kit was used for amplification. The quality of the melting curve was checked, and the Ct value was obtained for quantitative analysis and statistics. The detection results are as Figure 5 shown. Figure 5 The abscissa in [figure] is the concentration of cinobufotalin. Figure 5 In [figure], the relative mRNA levels of cell cycle-related genes CCNA2, CCNB1, and CDK1 decreased, while the expression level of p16 increased. In addition, the expression level of the typical proliferation marker Ki67 also decreased significantly. These results indicate that cinobufotalin can induce cell cycle arrest and reduce the proliferation ability of tuberous sclerosis complex-related renal angiomyolipoma cells.

[0054] The present invention has been described in detail above in combination with specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art understand that without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements can be made to the technical solutions and their implementation manners of the present invention, and these all fall within the scope of the present invention. The protection scope of the present invention is subject to the appended claims.

Claims

1. Application of cinobufotalin in the preparation of a drug for treating tuberous sclerosis complex-related renal angiomyolipoma; The chemical structural formula of the cinobufotalin is shown in formula (1): Formula (1).

2. The application according to claim 1, wherein the cinobufotalin can inhibit the growth of tuberous sclerosis complex-related renal angiomyolipoma cells.

3. The application according to claim 1, wherein the cinobufotalin can increase the protein expression levels of Caspas3, Caspas9, Cytochrome C, and γ-H2A.X in tuberous sclerosis complex-related renal angiomyolipoma cells.

4. The application according to claim 1, wherein the cinobufotalin can reduce the protein expression level of CCNA2 in tuberous sclerosis complex-related renal angiomyolipoma cells, and the cinobufotalin can increase the protein expression level of p16 in tuberous sclerosis complex-related renal angiomyolipoma cells.

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